High‐level seroprevalence against Leptospira interrogans serovars among wild foxes, jackals and stray dogs in the North Khorasan Province, Iran

Abstract Background Leptospirosis is an important, neglected zoonotic disease that affects people and animals in humid (sub)tropical regions. Wild canines carry the pathogen and may contaminate natural resources which may then act as a source of human infection. Objectives The study was designed to understand the seroprevalence of leptospirosis among domestic and wild canines in Bojnurd County, Northeast Iran. Methods A total of 77 serum samples, comprising 29 sera from asymptomatic wild canines [foxes (n = 25) and jackals (n = 4)] and 48 sera from asymptomatic stray dogs, was investigated. Serovars were identified and antibody titres were measured by standard microscopic agglutination test (MAT) using serial serum dilutions. Results Among all serum samples, 44.1% reacted positively to a Leptospira interrogans serovars. The average percentage of positive reactions was higher in stray dogs than in wild canines although this did not reach statistical significance (55.2% and 37.5%, p = 0.159). Positive reactions with L. i. Pomona, L. i. Australis and L. i. Tarasovi was detected only among jackals and foxes. Among the stray dogs, the highest number of positive sera were for L. i. Grippotyphosa (61.1%) and L. i. Canicola (50%). The highest titre detected was for L. i. canicola (1:1600) in two stray dogs and against L. i. Icterohaemorrhagiae and L. i. Pomona (1:800) in a single jackal. Conclusions The study revealed that leptospirosis is endemic among various canine species in the North Khorasan Province of Iran. Detailed monitoring of canines is necessary for better understanding the epidemiology of infection in our and other Iranian regions.


INTRODUCTION
Leptospirosis, a globally neglected zoonotic disease causing fever for days to weeks, affects people and animals in humid (sub-)tropical regions (Ullmann & Langoni, 2011). Leptospirosis is mild in 90% of all cases but may generate severe complications in other patients.
Although the main symptoms are fever, muscle pain and headaches, the disease can also lead to serious organ failure (kidneys, liver) and haemorrhaging. The disease is spread by a large variety of both wild and domestic animals which are natural reservoirs of Leptospira spp. (Adler & de la Peña Moctezuma, 2010). A wide variety of animals host Leptospira interrogans and many of these are asymptomatic Leptospira renal carriers. They contaminate the environment by shedding bacteria in their urine and they may develop symptoms only after long incubation periods (Adler & de la Peña Moctezuma, 2010). The precise epidemiology of leptospirosis in a specific niche is defined by the close contact between the particular Leptospira serovars and their specific maintenance hosts (Fratini et al., 2020). The seroprevalence of leptospirosis in humans and different animals in Iran has been studied before (Khalili et al., 2020). Leptospirosis is endemic in the North Khorasan province, as recent studies using the microscopic agglutination test (MAT) unveiled past and present infections among both rodents (Arzamani et al., 2018) and humans (Hashemi et al., 2021). Leptospiral infection, its associated prevalence and the dominant serovars were reported as being different in canines around the world (Ab Rahman et al., 2018;Ambily et al., 2013;Aslantaş et al., 2005;Azocar-Aedo et al., 2017;Lelu et al., 2015;Samir et al., 2015;Shi et al., 2012). Similar reports on seroprevalence have emerged from different geographic regions of Iran as well (Avizeh et al., 2009;Fahimipour et al., 2021;Jamshidi et al., 2008;Rad et al., 2004;Torkan & Momtaz, 2019

Study population and blood sample collection
This study was conducted in the North Khorasan province in the Northeast of Iran (37.47 N and 57.33 E) (Figure 1). In a six months period, from April to September in 2020, stray dogs and wild canines were collected by the municipal animal control department and examined by veterinarians. All asymptomatic animals subjected to this study. A total of 77 blood samples were randomly selected from 29 asymptomatic wild canines [foxes (n = 25) and jackals (n = 4)] and 48 also randomly selected asymptomatic stray dogs were included in the current study.
Five millilitres of blood were collected aseptically from each animal and centrifuged at 3000 rpm for 10 min. Sera were kept at -20 • C in micro-tubes. The sera were transferred to the Leptospira Research

Microscopic agglutination test (MAT)
All serum samples were subjected to MAT in duplicate to determine the exposure of individual animals to the various serovars of L. interrogans bacteria (Niloofa et al., 2015;Sakhaee, 2011;Sakhaee et al., 2010). A seven to 10 days' culture of different serovars of L. interrogans in a liquid medium (GRA-Sina, Sinajen, Tehran, Iran) was used as a source of cellular antigens. The density of leptospires was checked using a counting chamber (Petroff-Hauser USA) and adjusted to 2 × 10 8 cell/ml. All

Statistical analysis
Fisher exact testing was conducted for comparing the leptospiral seroprevalence among the different animals using SPSS software (version 20).

RESULTS
Among the 77 samples studied, 44.1% reacted positively to at least one serovar-specific antigen preparation of L. interrogans. The rate of positive reactions was higher in stray dogs versus wild canines (55.2% and 37.5%), but this was not statistically significant (p = 0.159). The highest detected frequency was for L. interrogans serovar Grippotyphosa (n = 14) and L. interrogans serovar Canicola (n = 11) ( Table 1).

DISCUSSION
Leptospirosis is one of the most significant re-emerging infectious diseases in Iran (Parhizgari et al., 2017). The present study investigated the prevalence of antibodies against serovar-specific leptospiral antigens among wild canines and stray dogs using the MAT (Niloofa et al., 2015). Clinical samples were collected from animals captured in the North Khorasan province in Iran. The location of the study is in a mountainous area with 9 months of cold weather. Conditions such as these should always be considered in our type of study but the precise impact of climate is ill defined. We report specific antibodies against various L. interrogans serovars found in jackals and foxes. The prevalence of specific antibodies against those leptospiral serovars detected among foxes in this study (55.2%)

TA B L E 2 Prevalence and the most dominant leptospiral serovars reported from Iran and other region of world among dogs
is higher than the prevalence reported in native foxes in Chile (7.7%) (Galarce et al., 2021), red foxes from Spain (47.1%) (Millán et al., 2009) and Croatia (33.8% and 31.25%) (Slavica et al., 2008;Slavica et al., 2011). The prevalence was slightly lower than recorded among red foxes from Croatia (57.6%) (Milas et al., 2006). In a recently conducted study on jackals, Nakonechnyi et al. (2019) found positive reactions against Leptospira interrogans among all nine animals that were tested.
The documented overall prevalence of 37.5% among dogs in this study is almost 2.5-fold higher than the estimated overall prevalence of 14.6% (95% CI: 3.49-25.77) for dogs in Iran (Khalili et al., 2020).
Based on serological data collected during the current study, we conclude that dogs, foxes and jackals are natural reservoirs of Leptospira in the area that we covered. Specific antibodies against L.
interrogans were reported as was done among rodents in the same geographical regions before (Arzamani et al., 2018). Dogs are considered an important host for Leptospira interrogans, and having close contact with stray dogs may increase the risk of infection in humans (Lelu et al., 2015).

CONCLUSION
We demonstrate that leptospirosis is a significant endemic epizootic disease in the study region. More extensive investigations on the large population of wild canines is recommended for better understanding the possible transfer of infections from wild and domesticated animals to humans. First thing to better substantiate this risk would be to perform seroprevalence studies among humans from which in the end prophylactic measures may be designed.

CONFLICT OF INTEREST
The authors have no relevant financial or non-financial interests to disclose.

FUNDING INFORMATION
The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

ETHICS STATEMENT
Sample collection was performed according to the rules and regula-

DATA AVAILABILITY STATEMENT
Data openly available in a public repository that issues datasets with DOIs.